1. Detection of Peroxide. Various methods are known for detecting hydrogen peroxide and related peroxides.
a. Colorimetric Detection of Peroxide. Compounds which react with hydrogen peroxide and a peroxidase to produce a colored product include ABTS, 4-aminoantipyrine (Anal. Letters, 26, 87 (1993)), and leuco dyes such as Leucocrystal Violet. Numerous methods exist for detecting hydrogen peroxide using a chromogen and a transition metal compound.
b. Fluorescent Detection of Peroxide. Compounds which react with hydrogen peroxide and a peroxidase to produce a fluorescent product include 2′,7′-dichlorofluorescin, dihydrorhodamine 123 (Arch. Biochem. Biophys., 302(2), 348-55 (1993)) and N-acetylresorufin, (Chemical & Pharmaceutical Bulletin, 49(3), 294-29, (2001)).
c. Chemiluminescent Detection of Peroxide. Acridinium esters and sulfonamides undergo a rapid oxidation reaction with hydrogen peroxide at alkaline pH to produce a flash of chemiluminescence (e.g. U.S. Pat. Nos. 4,745,181, 4,946,958, 5,281,712 and 5,468,646). Lucigenin (9,9′-biacridinium dinitrate) is oxidized by hydrogen peroxide to produce chemiluminescence (Maskiewicz, et al., J. Am. Chem. Soc., 101, 5347-5354 (1979)).
Esters and amides of oxalic acid react with hydrogen peroxide in the presence of a fluorescer to produce chemiluminescence. This reaction formed the basis of the well-known “light stick” technology used in novelty items.
Cyclic acylhydrazides including the amine-substituted compounds luminol and isoluminol, hydroxy-substituted compounds and heterocyclic analogs react with hydrogen peroxide and a metal catalyst to produce chemiluminescence. Metal catalysts include heme, hexacyanoferrate and other transition metal ions including Cu(II) and Co(II).
U.S. Pat. No. 5,545,834 describes the chemiluminescent reaction of spiro-acridan compounds with hydrogen peroxide. The reaction is enhanced by the addition of horseradish peroxidase.
d. Enzymatic Detection of Peroxide. Various reagents have been developed for detection of peroxidase activity by reaction of a peroxidase enzyme, a source of hydrogen peroxide and an indicating reagent. These reagents therefore also serve to detect hydrogen peroxide. Color, fluorescence or chemiluminescence can be produced with use of the appropriate reagent. Chemiluminescent substrates include amino-substituted cyclic acylhydrazides such as the well-known luminol and isoluminol (Anal. Chim. Acta, 170, 101-107, (1985)), heterocyclic acylhydrazides (M. Ii, et al., Biochem. Biophys. Res. Comm., 193(2), 540-5 (1993); U.S. Pat. No. 5,324,835 and Y. Tominaga, et al., Tetrahedron Lett., 36, 8641-4 (1995)), and hydroxy-substituted phthalhydrazides (U.S. Pat. No. 5,552,298).
Applicant's U.S. Pat. Nos. 5,491,072, 5,523,212 and 5,593,845 disclose chemiluminescent N-alkylacridan-carboxylic acid derivatives which produce light upon reaction with a peroxide and a peroxidase. Applicant's U.S. Pat. No. 5,922,558 discloses a class of compounds containing an electron-rich double bond as chemiluminescent peroxidase substrates.
European Patent Specification EP0682254B1 discloses assay methods in which a conjugate of an enzyme that generates hydrogen peroxide is used and the peroxide is detected by acridinium ester chemiluminescence.
Fluorescent substrates for peroxidase include 3-(4-hydroxyphenyl)propionic acid as disclosed in U.S. Pat. No. 6,040,150, 2-(4-hydroxyphenyl)acetic acid disclosed in Zaitsu and Ohkura, Anal. Biochem., 109, 109-113 (1980), homovanillic acid and tyramine (Y. Li, et al., Anal. Chim. Acta, (340), 159-168, (1997)), o-phenylenediamine and N,N′-dicyanomethyl-o-phenylenediamine (Li, et al., Microchem. J., 53(4), 428-436 (1996)), amide and carbamate derivatives of p-aminophenol (M. Kawaguchi, et al., Bioluminescence and Chemiluminescence Perspectives for the 21st Century, A. Roda et al., Eds., Wiley & Sons, Chichester, pp 508-511, (1999)), 3,4-dihydro-2(1H)-quinoxalone and related derivatives (Li, et al., Anal. Chim. Acta, 340(1-3), 159-168 (1997)), reduced forms of fluorescein, rhodamine and other xanthine dyes and fluorinated derivatives of the latter (U.S. Pat. No. 6,162,931).
Chromogenic or color-forming substrates for peroxidase include tetramethylbenzidine, chlorophenol red and 2,2′-Azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid (Ngo, T. T. In Immunochemistry of Solid-Phase Immunoassay, Butler, J. E. Ed., CRC: Boca Raton, 1991, pp 85-102.).
2. Fluorescent and Colored Boronic Acid Sensors.
Fluorescent and colored compounds containing boronic acid substituents for use in detection methods are disclosed in U.S. Pat. Nos. 4,496,722, and 4,659,817. The boronic acid group complexing compounds coordinate to and bind pairs of hydroxy, amine or thiol groups to form a fluorescent or colored complex. The binding partner can be either a carrier compound such as a buffer salt or it can be a biological substance which is to be tagged. Representative of the latter are cellular components. These methods differ fundamentally from the methods of the present invention by virtue of the unbound boronic acid compound being already colored or fluorescent to the same extent as the bound complex with the carrier or cellular component. Color or fluorescence is not created during the conduct of the methods.
The '772 and '817 patents also disclose peroxide assays in which the boronic acid substituted fluorescent or colored compounds are reacted with hydrogen peroxide to cleave the fluorescent or colored reporter moiety from the boronic acid group. Detection requires a separation step in order to measure the liberated reporter. Organic solvent extraction, release from a solid phase and filtration are disclosed as means to separate the released reporter from the reporter-boronic acid compound. Again, in contrast to the methods of the present invention, color or fluorescence is not created during the conduct of the methods.
Colored or fluorescent boronic acid complexing agents for use in detection of glycated blood proteins such as hemoglobin are disclosed in U.S. Pat. Nos. 5,242,842, 5,506,144 and 5,739,318. The boronic acid groups coordinate to and bind pairs of hydroxy groups to form a colored or fluorescent complex. The complex is separated from unbound complexing agent and measured. Detection does not involve any peroxide. This mode of measurement differs from the present invention in requiring a separation and that the unbound boronic acid compound is already colored or fluorescent to the same extent as the bound protein complex.
PCT Publication WO 02/46752 discloses assays for polyhydroxyl compounds, e.g. glucose, using boronic acid-quencher conjugates which bind to and quench the fluorescence of appropriately substituted fluorescers. Binding of the fluorescer via the boronic acid group of the quencher is reversed in the presence of the polyhydroxyl compound which competes for binding with the boronic acid group.
3. Phenylboronic acid Peroxidase Enhancers.
U.S. Pat. Nos. 5,512,451 and 5,629,168 disclose phenylboronic acid compounds as enhancers of the peroxidase-catalyzed chemiluminescent oxidation of luminol with hydrogen peroxide. In the methods disclosed therein the boronic acid compound promotes the reaction of the peroxidase in oxidizing luminol. Chemiluminescence is produced from an oxidized form of luminol and not from the boronic acid. Arylboronic acid derivatives are disclosed as peroxidase enhancers in the chemiluminescent oxidations of acridan compounds in U.S. Pat. Nos. 5,723,295 and 5,922,558.
None of the foregoing methods disclose the use of boronic acid or boronate ester signalling compounds in a method for detecting and, when desired, quantifying the amount of hydrogen peroxide wherein the peroxide causes the formation of a detectable signal from the precursor signalling compound which does not itself possess the property being detected.